The present invention relates to a method and an agent, and also their use, for the stabilization of alkaline phosphatase in buffer solutions, e.g., as they are used in clinical chemistry for determining the enzymatic activity of alkaline phosphatase and its isoenzymes.
The determination of the enzymatic activity of alkaline phosphatase (EC 3.1.3.1) in serum or plasma plays an important part in clinical diagnosis. This determination is carried out in buffered aqueous solution and the pH of the solution should be above 8. At first, barbitone buffer (pH 8.6), and later carbonate/bicarbonate buffer (pH 9.9) or glycine/sodium hydroxide solution buffer (pH 10.5), were used. After it had been found that high concentrations of aminoalcohols, which can act as phosphoryl acceptors, increase the enzymatic activity of alkaline phosphatase, diethanolamine and 2-amino-2-methylpropan-1-ol, in particular, were employed as buffer substances in determinations of the enzymatic activity of alkaline phosphatase in clinical chemistry. However, these buffer substances are disadvantageous since they can contain impurities which inhibit or inactivate alkaline phosphatase. For diethanolamine, the impurity is ethanolamine, which can be separated off by expensive rectification. In the case of 2-amino-2-methylpropan-1-ol, a substituted ethylene derivative has been identified from a number of impurities as an inactivator of alkaline phosphatase [Clin. Chem. 24, 1,611 (1978)]. Its structure has not yet been completely elucidated.
It is known from Clin. Chim. Acta 98, 61 (1979) that the inactivation of alkaline phosphatase in 2-amino-2-methylpropan-1-ol buffer can be avoided if zinc salts are added. However, this selection is highly disadvantageous since, for concentrations of zinc ions which are greater than the optimum concentration, the alkaline phosphatase is again inhibited. Alkaline phosphatase is guaranteed to have maximum enzyme activity only if an exact dose of the zinc salts is added. This optimum concentration of added zinc ions, however, is difficult for each batch of buffer.